COMPARATIVE STUDY BETWEEN LONG AND SHORT TERM ESTROUS SYNCHRONIZATION PROTOCOLS ON ESTROUS AND FERTILITY RESPONSES OF CYCLIC EWES

ABD-EL-RAHMAN, HOWIDA; IBRAHIM, MAHA

doi:10.21608/avmj.2018.168963

COMPARATIVE STUDY BETWEEN LONG AND SHORT TERM ESTROUS SYNCHRONIZATION PROTOCOLS ON ESTROUS AND FERTILITY RESPONSES OF CYCLIC EWES

Document Type : Research article

Authors

HOWIDA ABD-EL-RAHMAN ¹
MAHA IBRAHIM ²

¹ Field Investigation Department, Animal Reproduction Research Institute, Giza, Egypt

² Biology of Reproduction Department, Animal Reproduction, Research Institute (ARRI), Giza, Egypt

10.21608/avmj.2018.168963

Abstract

The present study was undertaken to minimize the period over which the progesterone impregnated sponges used for estrous synchronization protocols from 12 days to only 7 days, for improving conception rate and assess the serum hormonal concentrations, protein profile changes as well as oxidant- antioxidant status to compare short term with long term protocol in cyclic Barki ewes. Twenty multiparous cycling ewes (2 - 4 years old) were randomly assigned into two equal groups (10 each), the first group was the short term protocol group (S-term protocol) and treated with sponges containing 50 mg medroxy progesterone acetate (MAP) for 7days. The second group was the long term protocol group (L-term protocol), also treated with the same sponges but for 12 days. At the time of sponge withdrawal, all ewes were intramuscularly injected with 200 IU PMSG (Pregnant Mare Serum Gonadotropin). Four fertile rams were introduced to all the ewes in the two groups for estrus detection and natural mating on the day of sponge withdrawal. Blood samples were collected on day of sponge insertion; on the 4^th day, the 6^th day of insertion; on the day of sponge withdrawal; on the 2^nd day of withdrawal as well as at one month after withdrawal from both groups. Conception rate as well as lambing rate were significantly higher (P>0.05) in S-term protocol (90%) than in L-term protocol group (60%). Serum progesterone level was significantly (P<0.05) higher in S-term protocol than L-term protocol group on day of withdrawal and during the first month after withdrawal (1.97 ± 0.08 vs 1.34 ± 0.10 ng/ml) and (2.78 ± 0.15 vs 1.89 ± 0.17 ng/ml), respectively. There was a significant decrease (P>0.05) in MDA level on the 2^nd day of withdrawal and at one month after withdrawal in S-term protocol ewes than L-term protocol (10.17± 0.25, 9.02± 0.22 and 12.45±0.54, 9.75±0.22 nmol\ml)), respectively. Short-term progestagen protocol (7 days) for estrus synchronization resulted in an improved fertility than the common long-term protocol (12 days) in adult cyclic ewes.

Keywords

Full Text

Assiut University web-site: www.aun.edu.eg

COMPARATIVE STUDY BETWEEN LONG AND SHORT TERM ESTROUS SYNCHRONIZATION PROTOCOLS ON ESTROUS AND FERTILITY

RESPONSES OF CYCLIC EWES

HOWIDA M.A. ABD-EL-RAHMAN ¹ and MAHA A. IBRAHIM ²

¹ Field Investigation Department, Animal Reproduction Research Institute, Giza, Egypt.

²Biology of Reproduction Department, Animal Reproduction Research Institute, Giza, Egypt.

Received: 28 June 2018; Accepted: 30 July 2018

ABSTRACT

Key words: Synchronization, progestagen, protein, antioxidants, ewes.

INTRODUCTION

The estrus synchronization technique is an important management technique that has been used to improve the reproductive efficiency of sheep and goats (Abecia et al., 2012). Progestagens are widely used to synchronize estrus (Evans, 2001). Intravaginal sponge impregnated with progestogens being the most commonly used for estrus synchronization in sheep (Manes et al., 2010 and Abecia et al., 2012), as it provide estrus synchronizaion by extending the luteal phase during the treatment period in ewes (Wildeus, 1999; Whitley and Jackson 2004). The common applied progestagen treatment for oestrous synchronization in ewes lasts 12-14 days, a time period similar to the lifespan of a cyclic corpus luteum

Corresponding author: Dr. MAHA A. IBRAHIM

E-mail address: maha_doctor2013@yahoo.com

Present address: Biology of Reproduction Department, Animal Reproduction Research Institute, Giza, Egypt.

(Abecia et al., 2012), followed by intramuscular (IM) injection of equine Chorionic Gonadotropin (eCG) appear to be the most practical method for estrus synchronization in ewes (Gomez et al., 2006; Swelum et al., 2015). However, this common treatment with a prolonged time of administration has been found to have detrimental effects on conception rate (Martin et al., 2004) due to ovulation of aged follicles (Johnson et al., 1996; Viñoles et al., 2001).

So, as long time progesterone applications have had suppressive effects on fertility and thus short period progesterone applications have been suggested as an alternative (Vinoles et al., 2001; Ali, 2007; Husein et al., 2007), because short-term protocols possibly allow for facilitating the managerial tasks, minimizing the vaginal discharge and infection risks and thus increasing the fertility rates. During different physiological periods, protein as a nutrient is an essential component for the female during her life; Serum protein is essential biochemical parameter for reproduction because it plays major role in embryogenesis and gametogenesis. (McBurney et al., 2002).

Oxidative stress alters the endocrine status, duration of estrus, follicular growth and development, and early embryonic development, all of which have detrimental effects on fertility (Fuquay, 1981; Dobson et al., 2012). The impacts of oxidative stress on the reproductive efficiency of live-stock are well documented (Ayo et al., 1996; Dobson et al., 2012). Using exogenous progesterone during synchronization increases lipid oxidation (Sönmez et al., 2009).

Our goal was to minimize the period over which progestagen sponges are inserted, from 12 days to only 7days to produce good results in establishing oestrus and improving conception rate and assess the serum hormonal concentrations. Protein profile changes and oxidant- antioxidant status on different days of the synchronization were followed up during both the short and the long term synchronization protocols for Barki ewes in breeding season.

MATERIALS AND METHODS:

The present study was performed at the experimental farm of Animal Reproduction Research Institute (ARRI). Twenty multiparous cycling Barki ewes (2 - 4 years old) were selected randomly from the flock and used in the present experiment. All ewes were healthy and clinically free from external and internal parasites. Animals housed in semi open pens under natural day light and temperature.

According to standard farming practice of ARRI, the animals were fed twice a day and had free access to drinking water and mineral blocks. They were fed with good-quality Egyptian clover, barseem (alfa alfa) every day and were offered a standard total mixed ration according to NRC (2007).

Animal groups and treatment protocols:

Before the start of the experiment, ewes were examined by ultrasound scanner (200 pie Medical Co – Nertherland - Holand), to confirm that ewes were non-pregnant.

Ewes were randomly assigned to two equal groups (10 each), the estrous cycles of all ewes were synchronized as follow: the first group (Short term protocol group) S-term protocol group, treated with sponges containing 50 mg medroxy progesterone acetate (MAP) which inserted intravaginally for 7days. The second group (Long term protocol group) L-term protocol group, treated with the same sponges but for 12 days. All ewes were injected with 200 IU PMSG at the time of sponge withdrawal.

Estrus detection and mating:

Four fertile Barki rams with good body conditions score were introduced to the ewes, two for each experimental group for estrus detection and mating; starting on the sponge withdrawal day. Rams were allowed to rotate among different ewes in the two groups to avoid sire/group confounding effect.

Measured traits throughout the experimental period:

Fertility was monitored in terms of conception rate, and lambing rate, all data was statistically analyzed using Costat program.

Conception rate =

number of conceived ewes / number of mated ewes x 100.

(Ozyurtlu et al., 2011)

Lambing rate =

number of lambing ewes / number of mated ewes x 100. (Zeleke et al., 2005)

Blood samples:

Blood samples were collected from the jugular vein in the early morning on day of insertion (day of sponge insertion); on the 4^th day, the 6^th day of insertion; on the day of withdrawal (day of sponge withdrawal); on the 2^nd day of withdrawal as well as at one month after withdrawal and ewes breeding from both groups. Whole blood was placed in a plain centrifuge tubes for serum separation. Serum was stored at -20°C till assay of progesterone, estradiol, and some biochemical parameters [total protein (TP), albumin (Alb.)] and antioxidant status parameters (glutathione peroxidase (GPX) and malondialdehyde (MDA).

Hormonal and biochemical analyses:

Hormones were assayed using ELISA kits (Monobind Inc. Lake forest, CA 92630, USA): progesterone (P4) Ross et al. (1981); estradiol (E17β) Ratcliffe et al. (1988). Total protein (TP) (Doumas et al., 1981), albumin (Doumas et al., 1971), and globulin was calculated by substracting the albumin values from the total protein values (Eckersall, 2008), glutathione peroxidase (GPX) Pagila and Valentine (1967) and Malondialdehyde (MDA) (Ohkawa et al., 1979) by colorimetric method.

Statistical analysis:

Results are expressed as mean ± standard error (SE). Differences between means in different groups were tested for significance using a one-way analysis of variance (ANOVA) followed by Duncan’s test, conception and lambing rates data were subjected to statistical analysis by Microstate copy right (c) 1984; Eco soft, Inc. employing a completely randomized design according to Snedecor and Cochran (1982).

RESULTS

Conception and lambing rates of S-term protocol and L-term protocol groups were represented in table (1), conception and lambing rates were significantly higher (P>0.05) in S-term protocol group than that in L-term protocol group (90% vs 60%), respectively.

Table 1: Conception and lambing rates of Barki ewes after synchronization by short and long term protocols (n=10).

Groups	Conception rate	Lambing rate
S – term	(9\10 ) 90%^a	(9\10) 90%^a
L – term	(6\10 ) 60%^b	(6\10) 60%^b

Data with different superscripts within the same column are significantly different at (P<0.05).

In the present study, the progesterone level in S-term protocol group, was significantly (P<0.05) increased after sponge insertion and reached peak level (6.00 ± 0.19 and 5.70 ± 0.22 ng/ml) on the 4^th and on the 6^th days of insertion after that it significantly (P<0.05) decreased to reach (1.97 ± 0.10 ng/ml) on the dayof withdrawal (day 7), and continued decreasing tell reached the minimal level on the 2^nd day of sponge withdrawal, after that it began to increase again during the first month after withdrawal. Similar trend was also noticed in ewes of L-term protocol group, and its levels were (5.86 ± 0.24; 5.67 ± 0.22; 1.30 ± 0.0; 0.40 ± 0.03 and 1.89 ± 0.16 ng/ml) on the 4^th and the 6^th days of insertion; dayof withdrawal (day 12); the 2^nd day of withdrawal and during the first month after withdrawal, respectively (Fig. 1).

Data with different small and capital letters are significantly different at (P<0.05)

Figure (1): Serum Progesterone levels in Barki ewes during synchronization by short and long term protocols (n=10).

It was found that serum progesterone level was significantly (P<0.05) higher in S-term protocol group than that in L-term protocol group on day of withdrawal and during the first month after withdrawal (1.97 ± 0.08 vs 1.34 ± 0.10 ng/ml) and (2.78 ± 0.15 vs 1.89 ± 0.17 ng/ml), respectively (Table 2).

Table 2: (mean ± SEM) of serum Progesterone levels (ng\ml) in Barki ewes during synchronization by short and long term protocols (n=10).

	Day of insertion	4^th day of insertion	6^th day of insertion	Day of withdrawal	2^nd day of withdrawal	One month of withdrawal
S – term	2.02 ± 0.07	5.65 ± 0.23	6.00 ± .19	1.97 ± 0.08^a	0.44 ± 0.06	2.78 ± 0.15^a
L- term	1.99 ± 0.07	5.67 ± 0.21	5.86 ± .24	1.34 ± 0.10^b	0.40 ± 0.04	1.89 ± 0.17^b

Data with different superscripts within the same column are significantly different at (P<0.05).

Data presented in Table (3), revealed that there was a non-significant difference between the pattern of serum estradiol level during estrus synchronization between S-term protocol and L-term protocol groups.

Table 3: (mean ± SEM) of serum Estradiol levels (pg\ml) in Barki ewes during synchronization by short and long term protocols (n=10).

	Day of insertion	4^th day of insertion	6^th day of insertion	Day of withdrawal	2^nd day of withdrawal	One month after withdrawal
S – term	4.63 ± 0.31	3.31 ± 0.19	3.63 ± 0.08	2.90 ± 0.19	17.03 ± 0.60	8.60 ± 0.44
L- term	5.21 ± 0.44	3.31 ± 0.17	3.28± 0.16±	2.60 ± 0.22	16.20 ± 0.79	7.24 ± .0.76

Tables (4, 5 and 6) showed serum levels of total protein, albumin and globulin in S-term protocol group as compared with L-term protocol group. There was non-significant difference between the two groups in all the experimental days in these parameters except in serum globulin level of L-term protocol group at one month after sponge withdrawal which was significantly (P<0.05) higher than that in S-term protocol group ewes.

Table 4: (mean ± SEM) of serum Total protein levels (mg\dl) in Barki ewes during synchronization by short and long term protocols (n=10).

	Day of insertion	4^th day of insertion	6^th day of insertion	Day of withdrawal	2nd day of withdrawal	One month after withdrawal
S – term	6.60 ± 0.10	6.70 ± 0.10	6.45 ± 0.13	6.65 ± 0.10	6.96 ± 0.13	6.45 ± 0.20
L- term	6.80 ± 0.16	6.60 ± 0.16	6.41 ± 0.16	6.96 ± 0.10	6.13 ± 0.10	6.70 ± 0.16

Table 5: (mean ± SEM) of Serum Albumin Levels (mg\dl) in Barki ewes during synchronization by short and long term protocols (n=10).

	Day of insertion	4^th day of insertion	6^th day of insertion	Day of withdrawal	2nd day of withdrawal	One month after withdrawal
S – term	3.78 ± 0.10	3.55 ± 0.13	3.42 ± 0.15	3.30 ± 0.13	3.18 ± 0.10	3.53 ± 0.10
L- term	3.85 ± 0.06	3.55 ± 0.10	3.30 ± 0.08	3.59 ± 0.13	3.28 ± 0.13	3.58 ± 0.10

Table 6: (mean ± SEM) of serum Globulinlevels (mg\dl) in Barki ewes during synchronization by short and long term protocols (n=10).

	Day of insertion	4^th day of insertion	6^th day of insertion	Day of withdrawal	2^nd day of withdrawal	One month after withdrawal
S – term	2.91 ± 0.16	3.21 ± 0.15	2.93 ± 0.93	3.35 ± 0.10	3.03 ± 0.14	2.50 ± 0.13^b
L- term	3.4 ± 0.19	2.95 ± 0.16	3.1± 0.18	3.57 ± 0.16	2.9 ± 0.19	3.24 ± 0.18^a

Data with different superscripts within the same column are significantly different at (P<0.05).

Serum GPX levels on the day of sponge insertion, 4^th day of insertion, 6^th day of insertion, day of sponge withdrawal, 2^nd day of withdrawal and after one month of withdrawal in S-term protocol group as compared to L-term protocol group were represented in Table (7). There were non-significance differences between the S-term protocol and L-term protocol groups in the different days of the trial.

Table 7: (mean ± SEM) of serum GPX levels (µM\ml) in Barki ewes during synchronization by short and long term protocols (n=10).

	Day of insertion	4^th day of insertion	6^th day of insertion	Day of withdrawal	2^nd day of withdrawal	One month after withdrawal
S - term	4.14 ± 0.25	4.45 ± 0.28	4.2 ± 0.25	3.88 ± 0.16	3.15 ± 0.09	6.65 ± 0.16
L – term	4.18 ± 0.22	4.05 ± 0.22	3.88 ± 0.25	4.02 ± 0.25	3.10 ± 0.16	6.47 ± 0.28

Serum MDA levels of S-term protocol and L-term protocol groups on the different experimental days were represented in Figure (2). It was observed that the lowest level of MDA of both experimental groups was on the day of sponge insertion which was significantly lower (P>0.05) than the other experimental days. In S-term protocol group MDA serum levels were significantly decreased (P>0.05) on the 2^nd day of sponge withdrawal and one month after sponge withdrawal than its levels on the 4^thand the 6^thdays of sponge insertion and on the day of sponge withdrawal. Meanwhile, at one month after sponge withdrawal its level did not differ significantly than that on the 2^nd day of sponge withdrawal and the day of sponge insertion. L-term protocol group showed significant (P>0.05) increase on the 2^nd day of sponge withdrawal than that on the day of sponge insertion, the 6^th day of insertion and after one month of sponge withdrawal. Meanwhile, there was non- significance difference between the 4^th day of sponge insertion and the day of sponge withdrawal. Also, there was non- significance difference between the 6^th day of insertion and at one month after withdrawal.

Data with different small and capital letters are significantly different at (P<0.05).

Figure (2): Serum malondialdehyde levels in Barki ewes during synchronization by short and long term protocols (n=10).

Serum MDA Levels on day of insertion and on the 4^th and the 6^th days of insertion; on the day of sponge withdrawal as well as at one month after sponge withdrawal between S-term protocol and L-term protocol groups were illustrated in Table (8), There was a significance decrease (P>0.05) in serum MDA level on 2^nd day of withdrawal and at one month after withdrawal in S-term protocol group ewes than that of L-term protocol group (9.75±0.25 ,12.45±0.54 and 9.02±0.22, 10.17±0.25), respectively. Meanwhile, there was non-significance different between the two groups in the other days of the experiment.

Table 8: (mean ± SEM) of Serum MDA levels in Barki ewes during synchronization by short and long term protocols (n=10).

	Day insertion	4^th day of insertion	6^th day of insertion	Day of withdrawal	2^nd day of withdrawal	One month after withdrawal
S – term	8.83 ± 0.41	11.13 ± 0.34	10.76 ± 0.28	11.27 ± 0.33	9.75 ± 0.25^b	9.02 ±.0.22^b
L- term	8.68 ± 0.25	11.01 ± 0.51	10.56 ±0.44	11.09 ± 0.47	12.45 ± 0.54^a	10.17 ±.0.25^a

Data with different superscripts within the same column are significantly different at (P<0.05).

DISCUSSION

With regard to serum P_4, in the present study, the difference in serum progesterone levels at day of sponge insertion before treatment between S-term protocol group and L-term protocol group were non-significant (2.02 vs 1.99 ng/ml), respectively; however, the present finding is lower than those reported by Mohan, (2017) in ewes; and Kusina et al. (2000) in goat which may be related to the different stages of the estrous cycle of the experimental animals used in these studies and our study. After initiation of experimental protocol there is a significant increase (P>0.05) in progesterone level in the two experimental groups till reach the peak level on the 4^th day till the 6^th day of sponge insertion, the present significant increase (P<0.05) might be due to the insertion of sponge which are resulted in sustained and slow release of progesterone during the period of its retention in vagina. Similar trend of increasing progesterone levels after vaginal sponge insertion were also reported by Hamra et al. (1986) who stated that progesterone concentration started to increase to near maximum levels within 24 h after sponge insertion in ewes synchronized with vaginal sponge, reached highest levels on day 4 and then declined. Kusina et al. (2000), also stated that plasma progesterone concentration increases sharply in does treated with intravaginal sponges, following insertion of sponge to reach (6.0-7.0 ng/ml) within 3-5 days after sponge insertion and this level are maintained thereafter until removal of sponge, then fall sharply to basal level after removal of sponges to reach a level which was less than (2.0 ng/ml) within 24 hrs. The elevation of progesterone level after insertion of vaginal sponge suggest that endogenous progesterone level were augmented indicating intravaginal delivery of progesterone via sponges cause effective and maintained elevated progesterone concentration during the duration of the experiment which is in accordance with the observation made by Kusina et al. (2000).

In sheep and goats, It was found that, the insertion of progesterone impregnated intravaginal device results in a rapid increase in blood progesterone concentrations within two days to reach (~5.0 ng/ml), and remain in a constant high level for approximately (4-5) days of treatment (Menchaca and Rubianes, 2004), similar to those levels observed during medium-late luteal phase. However, after 6 or 7 days of treatment, blood progesterone concentrations decrease gradually with time during the remaining period (Husein and kridi, 2002; Yavuzer, 2005) to sub-luteal levels (< 2.0 ng/mL), which enough for blocking ovulation but predisposing to a persistent growth of the dominant follicle, prolonged luteal function and reduced fertility (Johnson et al., 1996). If the intravaginal device is maintained during (12 or 14) days, the detrimental conditions of low progesterone levels described above are present during an excessive period, and then fertility is affected. For this reason, with the aim to promote the follicular turnover and thus allow the ovulation of a young follicle and a healthy oocyte with good fertility, the treatments for estrus synchronization should avoid the exposure to low levels of progesterone during an excessive period.

Progesterone level was significantly (P<0.05) decreased on the day of sponge withdrawal (7^th and 12^th day of sponge insertion) in S-term protocol and L-term protocol groups respectively, meanwhile, its level was significantly (P<0.05) higher in S-term protocol group than that in L-term protocol group on withdrawal day. After that it continued decreasing tell reach minimal level on the 2&

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